Ionic composition of the rectal contents and excreta of the reduviid bug Triatoma infestans

112

122

We cloned an aquaporin gene from Trypanosoma cruzi (TcAQP) that encodes a protein of 231 amino acids, which is highly hydrophobic. The protein has six putative transmembrane domains and the two signature motifs asparagine-proline-alanine (NPA) which have been shown, in other aquaporins, to be involved in the formation of an aqueous channel spanning the bilayer. TcAQP was sensitive to endo H treatment, suggesting that the protein is N-glycosylated. Oocytes of Xenopus laevis expressing TcAQP swelled under hyposmotic conditions indicating water permeability, which was abolished after preincubating oocytes with very low concentrations of the AQP inhibitors HgCl 2 and AgNO 3 . No glycerol transport was detected. Immunofluorescence microscopy of T. cruzi expressing GFP-TcAQP showed co-localization of TcAQP with the vacuolar proton pyrophosphatase (V-H ؉ -PPase), a marker of acidocalcisomes. This localization was confirmed by Western blotting and immunofluorescence staining using polyclonal antibodies against a C-terminal peptide of TcAQP. In addition, there was a strong anterior labeling in a vacuole, close to the flagellar pocket, that was distinct from the acidocalcisomes and that was identified by immunogold electron microscopy as the contractile vacuole complex. Taking together, the presence of an aquaporin in acidocalcisomes and the contractile vacuole complex of T. cruzi, provides support for the role of these organelles in osmotic adaptations of these parasites.

Section: Presence Of a Contractile Vacuole Complex In Different Life mentioning

confidence: 99%

A Functional Aquaporin Co-Localizes with the Vacuolar Proton Pyrophosphatase to Acidocalcisomes and the Contractile Vacuole Complex of Trypanosoma cruzi

Montalvetti

Rohloff

Docampo

2004

112

122

“…As T. cruzi passes through its digenetic life cycle, it encounters extreme fluctuations in external osmolarity. These osmotic fluctuations occur both within the insect gut (16) and also when the parasite moves through the vertebrate host. Previous studies on the response of T. cruzi to hyposmotic stress have shown that both insect and vertebrate stages possess a robust regulatory volume decrease (RVD) mechanism that completely reverses cell swelling (17).…”

mentioning

confidence: 99%

A Trypanosoma cruzi Phosphatidylinositol 3-Kinase (TcVps34) Is Involved in Osmoregulation and Receptor-mediated Endocytosis

Schoijet

Miranda

Girard-Dias

et al. 2008

Trypanosoma cruzi, the etiological agent of Chagas disease, has the ability to respond to a variety of environmental changes during its life cycle both in the insect vector and in the vertebrate host. Because regulation of transcription initiation seems to be nonfunctional in this parasite, it is important to investigate other regulatory mechanisms of adaptation. Regulatory mechanisms at the level of signal transduction pathways involving phosphoinositides are good candidates for this purpose. Here we report the identification of the first phosphatidylinositol 3-kinase (PI3K) in T. cruzi, with similarity with its yeast counterpart, Vps34p. TcVps34 specifically phosphorylates phosphatidylinositol to produce phosphatidylinositol 3-phosphate, thus confirming that it belongs to class III PI3K family. Overexpression of TcVps34 resulted in morphological and functional alterations related to vesicular trafficking. Although inhibition of TcVps34 with specific PI3K inhibitors, such as wortmannin and LY294,000, resulted in reduced regulatory volume decrease after hyposmotic stress, cells overexpressing this enzyme were resistant to these inhibitors. Furthermore, these cells were able to recover their original volume faster than wild type cells when they were submitted to severe hyposmotic stress. In addition, in TcVps34-overexpressing cells, the activities of vacuolar-H ؉ -ATPase and vacuolar H ؉ -pyrophosphatase were altered, suggesting defects in the acidification of intracellular compartments. Furthermore, receptor-mediated endocytosis was partially blocked although fluid phase endocytosis was not affected, confirming a function for TcVps34 in membrane trafficking. Taken together, these results strongly support that TcVps34 plays a prominent role in vital processes for T. cruzi survival such as osmoregulation, acidification, and vesicular trafficking.Phosphatidylinositol 3-kinase (PI3K) 6 activities have been found in all eukaryotic cell types examined to date (1, 2) and are linked to a diverse set of key cellular functions, including cell growth, survival, and intracellular trafficking. PI3Ks belong to a large family of enzymes that has been divided into three functional classes on the basis of their protein domain structure, lipid substrate specificity, and regulatory properties. Class I PI3Ks were the first ones to be identified and are important components of the signaling pathways that regulate eukaryotic cell growth (3, 4). These PI3Ks have a 110-kDa catalytic subunit that exhibits a substrate preference for PI 4-phosphate and PI 4,5-bisphosphate (5). Class II PI3Ks are less well known but may also function in the regulation of cell growth (5, 6) and, additionally, in clathrin-mediated endocytosis (7, 8). These enzymes prefer phosphatidylinositol (PI) as substrate but may also utilize PI 4-phosphate. Finally, class III family of PI3Ks is related to the yeast vacuolar protein sorting 34, Vps34p, and their homologs from other eukaryotes. Vps34p-like kinases specifically phosphorylate PI to produce phosphatidylinosi...

“…Extreme fluctuations in osmolarity occur within the gut of the vector (2,3), and also when the infective form of the parasite passes out of the vector in the highly concentrated excreta and rapidly encounters the interstitial fluid of the mammalian host with a much lower osmolarity.…”

mentioning

confidence: 99%

Acidocalcisomes and the Contractile Vacuole Complex Are Involved in Osmoregulation in Trypanosoma cruzi

Rohloff¹,

Montalvetti²,

Docampo

2004

114

149

Trypanosoma cruzi, the etiologic agent of Chagas disease, resists extreme fluctuations in osmolarity during its life cycle. T. cruzi possesses a robust regulatory volume decrease mechanism that completely reverses cell swelling when submitted to hypo-osmotic stress. The efflux of amino acids and K ؉ release could account for only part for this volume reversal. In this work we demonstrate that swelling of acidocalcisomes mediated by an aquaporin and microtubule-and cyclic AMP-mediated fusion of acidocalcisomes to the contractile vacuole complex with translocation of this aquaporin and the resulting water movement are responsible for the volume reversal not accounted for by efflux of osmolytes. Contractile vacuole bladders were isolated by subcellular fractionation in iodixanol gradients, showed a high concentration of basic amino acids and inorganic phosphate, and were able to transport protons in the presence of ATP or pyrophosphate. Taken together, these results strongly support a role for acidocalcisomes and the contractile vacuole complex in osmoregulation and identify a functional role for aquaporin in protozoal osmoregulation.